RESUMO
The safety and genetic stability of V4020, a novel Venezuelan Equine Encephalitis Virus (VEEV) vaccine based on the investigational VEEV TC-83 strain, was evaluated in mice. V4020 was generated from infectious DNA, contains a stabilizing mutation in the E2-120 glycoprotein, and includes rearrangement of structural genes. After intracranial inoculation (IC), replication of V4020 was more attenuated than TC-83, as documented by low clinical scores, inflammation, viral load in brain, and earlier viral clearance. During the first 9 days post-inoculation (DPI), genes involved in inflammation, cytokine signaling, adaptive immune responses, and apoptosis were upregulated in both groups. However, the magnitude of upregulation was greater in TC-83 than V4020 mice, and this pattern persisted till 13 DPI, while V4020 gene expression profiles declined to mock-infected levels. In addition, genetic markers of macrophages, DCs, and microglia were strongly upregulated in TC-83 mice. During five serial passages in the brain, less severe clinical manifestations and a lower viral load were observed in V4020 mice and all animals survived. In contrast, 13.3% of mice met euthanasia criteria during the passages in TC-83 group. At 2 DPI, RNA-Seq analysis of brain tissues revealed that V4020 mice had lower rates of mutations throughout five passages. A higher synonymous mutation ratio was observed in the nsP4 (RdRP) gene of TC-83 compared to V4020 mice. At 2 DPI, both viruses induced different expression profiles of host genes involved in neuro-regeneration. Taken together, these results provide evidence for the improved safety and genetic stability of the experimental V4020 VEEV vaccine in a murine model.
RESUMO
Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to the Americas. VEEV outbreaks occur periodically and cause encephalitis in both humans and equids. There are currently no therapeutics or vaccines for treatment of VEEV in humans. Our group has previously reported on the development of a benzamidine VEEV inhibitor, ML336, which shows potent antiviral activity in both in vitro and in vivo models of infection. In cell culture experiments, ML336 inhibits viral RNA synthesis when added 2-4 h post-infection, and mutations conferring resistance occur within the viral nonstructural proteins (nsP2 and nsP4). We hypothesized that ML336 targets an activity of the viral replicase complex and inhibits viral RNA synthesis. To test this hypothesis, we employed various biochemical and cellular assays. Using structural analogues of ML336, we demonstrate that the cellular antiviral activity of these compounds correlates with their inhibition of viral RNA synthesis. For instance, the IC50 of ML336 for VEEV RNA synthesis inhibition was determined as 1.1 nM, indicating potent anti-RNA synthesis activity in the low nanomolar range. While ML336 efficiently inhibited VEEV RNA synthesis, a much weaker effect was observed against the Old World alphavirus Chikungunya virus (IC50 > 4 µM), agreeing with previous data from a cell based assay. Using a tritium incorporation assay, we demonstrated that there was no significant inhibition of cellular transcription. With a combination of fluorography, strand-specific qRT-PCR, and tritium incorporation, we demonstrated that ML336 inhibits the synthesis of the positive sense genomic, negative sense template, and subgenomic RNAs of VEEV. Based on these results, we propose that the mechanism of action for this class of antiviral compounds is inhibition of viral RNA synthesis through interaction with the viral replicase complex.